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ISSN 0582-9879                                        ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(5): 547-552                                     CN 31-1300/Q

In vivo CTL Immunity Can be Elicited by OVA-linker-b2m Fusion Protein

QIAN Li, QIAN Guan-Xiang*

( Research Centre of Molecular Biology,  Shanghai Second Medical University,  Shanghai 200025,  China )

Abstract    To study the effect of OVA-linker-b2m fusion protein as an immunogen to induce CTL mediated immune response in mice. The b2m gene was amplified by PCR. OVA257-264 peptide sequence and an 8-amino acid linker were added to the COOH-terminus of b2m gene, and then cloned into pET-42b(+) vector. After that,  gene expression, protein purification and refolding were conducted. Then the protein product was used as an immunogen to induce an efficient CTL mediated immune response in vivo. Specific CTL responses were demonstrated by H-2Kb-OVA tetramer staining and cytotoxicity assay. Extracelluar IFN-g was also quantitatively analyzed. The results showed that specific CTL response could be induced in vivo by OVA-linker-b2m fusion protein. Generation of terameric peptide-MHC complexes in vitro is a powerful tool to stain specific CTL.

Key words    tetramer; gene expression; purification；refolding; CTL

Peptide-based vaccines should be safe and highly specific. However,  peptides often fail to prime CTL immunity in vivo[1] despite their ability to stimulate primary CTL responses and sensitize target cells in vitro[2, 3]. Most of these immunization protocols require the addition of incomplete Freund's adjuvant or other types of bacterially derived adjuvants[4],  as peptides alone rarely trigger an immune response or might even induce tolerance. Adjuvants are presumed to cause harmful side effects and therefore would not be acceptable for therapeutic practice. b2m protein has been observed to augment epitope-specific CTL responses in vivo when coinjected with peptide[5].This adjuvant effect was attributed to the ability of b2m to enhance peptide loading of surface class I molecules,  a well-defined in vitro phenomenon.We anticipated that a more potent immunogen could be generated if the peptide was covalently linked to b2m,  thereby restricting its diffusion and creating a molecule that contains two high affinity binding sites for class I heavy chains.

1  Materials and Methods

1.1  Plasmid, strain and cells

E.coli C600，HeLa-Kb,  L929-Kb cells were stored in our lab. E.coli BL21（DE3）and  pET-42b(+) were purchased from Novagen Co.. Purified H-2Kb and human b2m protein were constructed previously[6, 7].

1.2  Agents, mice

Restrictive endonucleases and T4 DNA ligase and PCR kit were purchased from Promega Co.. Peptides were synthesized at Genemed Synthesis Co., purified to >95%, homogeneity by reverse-phase HPLC. Mab 25-D1.16(anti-OVA257-264/Kb)was a kind  gift from Shubing Qian,  NIH. Mab(anti-hb2m),  Horseredish peroxidase(HRP) labeled  goat anti-mouse IgG antibody were purchased from Boshide Co.,  China. Six-to 8-wk-old female C57BL/6(H-2b) mice were purchased from Animal Center of the Chinese Academy of Sciences,  Shanghai.

1.3  Construction of peptide-MHC tetramers

The tetramer was made mainly according to the protocol of Altman et al[8]. There are some modifications based on the protocol. Briefly,  human b2m and soluble domain of the Kb heavy chain (residues 1-280) linked at its carboxyl terminus to a BirA substrate peptide were expressed separately in E.coli strain BL21(DE3) under the induction of IPTG (1 mmol/L). One liter of cells were collected by centrifugation and were lysed by sonication. The inclusion body pellet was dissolved in 4 ml of 8 mol/L urea / 0.1 mol/L NaH2PO4/ 0.01 mol/L Tris-HCl,  pH 8.0,  and insoluble material was pelleted by centrifugation at 15 000 g. The soluble protein was further purified by affinity chromatography. Refolding and complex formation were initiated by dilution of the two denatured subunits and peptide into 200 ml of 100 mmol/L Tris-HCl,  pH 8.0 / 400 mmol/L L-arginine·HCl / 2 mmol/L EDTA / 5 mmol/L reduced glutathione / 0.5 mmol/L oxidized glutathione / 0.5 mmol/L phenylmethylsulfonyl fluoride. The final concentrations of the heavy chain,  b2m,  and the peptide were 31 mg/L (1 mmol/L),  24 mg/L (2 mmol/L),  10mg/L (10 mmol/L),  respectively. The refolding mixture was incubated at 4 ℃ for 48 h. The 200 ml of refolding mixture was concentrated with PEG (MW: 12 000). The Kb-OVA complex was further purified on a source 15Q (Pharmacia) in 20 mmol/L Tris-HCl (pH 8.0),  with a gradient of NaCl from 0 to 0.5 mol/L. The purified proteins were stored in phosphate-buffered saline (PBS) plus a cocktail of protease inhibitors: 0.7 mg/L pepstatin; 2 mmol/L phenylmethysul-fonylfluoride; and 1 mmol/L EDTA. The folded material was then subjected to enzymatic biotinylation by BirA enzyme (Avidity Co.) at 30 ℃ for 30 min. The tetrameric complexes of biotinylated H-2Kb/peptide were produced by mixing purified,  biotinylated heterodimer with phycoerythrin-labeled Streptavidin (Sigma Co.) at a molar ratio of 4∶1.

1.4  Monoclonal antibody binding

To detect the conformation of the H-2Kb-OVA complex by ELISA, monoclonal antibody 25-D1.16(anti-OVA257-264/Kb, 1 mg/L) was used as the first antibody,  Horseredish peroxidase (HRP) labeled goat anti-mouse IgG antibody (1∶1 000 dilution) was used as the second antibody.    

1.5  Vector construction and protein expression

To construct a fusion gene for the described OVA-linker-b2m, the OVA (257-264)peptide sequence and the linker sequence were fused at the 5′end of b2m cDNA  sequence. The amplification of fusion gene was carried out by using PCR in a reaction mixture of 50 ml,  via 35 cycles of denaturation (94 ℃,  1 min),  annealing (55 ℃, 1.5 min), and extension(72 ℃, 1 min).The sequences of primers were as follows: 5′-cagcatatgtccataat-caactttgaaaaactcggaaggaggatccgaggtggcagcatccagcgtac-tccaaag-3′；  and 5′-caactcgagcatgtctcgatcccac-3′. The fusion gene was cloned into pET-42b(+) plasmid by NdeI and XhoI and transformed into the E.coli strain BL21 (DE3). The recombinant plasmid was sequenced. After induction of expression by adding IPTG to a final concentration of 1 mmol/L at 37 ℃ for 3 h,  cells were harvested by centrifugation at 4 000 g for 10 min,  then cells were lysed by ultrasonication and suspended into buffer B (8 mol/L urea,  0.1 mol/L NaH2PO4  / 0.01 mol/L Tris-HCl,  pH 8.0). The proteins were run on SDS-PAGE (12%). Subsequently,   the separated proteins were transferred onto NC membrane at 200 mA for 2 h. The membrane was incubated with mouse anti-human b2m monoclonal antibody (1∶200),  followed by an AP-conjugated second antibody.

1.6  Batch purification of His6 fusion protein

One ml of the 50% NiNTA slurry and 4 ml lysate were mixed gently,  then the  lysate-resin mixture was loaded into an empty column. The column was washed twice with 4 ml buffer C (8 mol/L urea / 0.1 mol/L NaH2PO4  / 0.01 mol/L Tris-HCl,  pH 6.3),  the recombinant proteins were eluted 4 times with 0.5 ml buffer D (same with buffer C except pH 5.9),  followed by 4 times with 0.5 ml buffer E (same with buffer C except pH 4.5). Fractions were collected and analyzed by SDS-PAGE. Proteins were refolded by dialysis against 5 mmol/L GSH / 2 mmol/L GSSG / 5 mmol/L EDTA / 50 mmol/L Tris-HCl,  pH 8.5. Then,  protein concen-tration was detected by Bradford method and the percentage of objective proteins was analyzed by gray scanning.

1.7  Detecting specific epitope density on cell surface

After incubating the fusion protein OVA-linker-b2m （10 mmol/L） with L929-Kb,  HeLa-Kb cell at 37 ℃ for 17 h,  the cells were harvested and stained with the first antibody 25-D1.16, followed by FITC-conjugated goat anti-mouse IgG as the second antibody. Stained cells were analyzed by FACS.

1.8  Induction of specific CTL in vivo and CTL detection

C57BL/6 mice were immunized with OVA peptide (100 mg), OVA peptide (2 mg)+ b2m (100 mg), OVA-linker-b2m (100 mg) by s.c.;  1 week later， splenocytes of the mice were separated and restimulated with OVA peptide（2 mmol/L）， rhIL-2 (50 u/ml); 5 days later， harvested cells were used as effector cells， the cytotoxicity assayed by quanti-tatively measuring lactate dehydrogenase (LDH) kit (Promega Co.),  the irradiated (3000 rads) L929-Kb cells were paused with OVA peptide (2 mmol/L) for 5 h as target cells. The extracellular IFN-g was quantitatively analyzed by ELISA kit (Promega Co.). Furthermore,  tetramer staining was performed as described reference[9]. In brief,  1 × 106 spleno-cytes were incubated in 100 ml of FACS buffer with 5-10 mg/L of PE-tetramer at 37 ℃ for 2 h. Cells were washed  and subsequently incubated with FITC labeled anti-CD8 at 4 ℃ for 30 min. All cells were washed twice after being stained with 2 ml PBS / 1% BSA before fixation in 1% formaldhyde. Stained cells were analyzed by FACS.   

2  Results

2.1  Detection of the conformation of H-2Kb-OVA complex by ELISA

We used the monoclonal antibody 25-D1.16 (anti-OVA257-264/Kb) to detect the conformation of H-2Kb-OVA complex by ELISA (Fig.1). Paired t test indicated that the data from ELISA for analyzing the differences between the refolded product and unrefolded product was significant (P<0.05). The Kb-OVA complex was further purified on a source 15Q (Pharmacia) in 20 mmol/L Tris-HCl (pH 8.0),  with a gradient of NaCl from 0 to 0.5 mol/L. Three peaks were seen in reconstitution experiments. Silver stain was performed to analyse the products in different peak fractons (Fig.2). The results showed that the purified Kb-OVA complex was in the first peak,  for there were two clear bands in this peak fraction. Peak 1 was collected and concentrated to 1 g/L. Tetrameric complexes of biotinylated H-2Kb/peptide was produced by mixing purified,  biotinylated heterodimer with phycoerythrin-labeled streptavidin at a molar ratio of 4∶1.

Fig.1  The result from ELISA

●, refolded product;  ○, unrefolded product.

Fig.2  The results from purification, silver staining

2.2  Identification of pET-OVA-linker-b2m recom-binant plasmid  

The recombinant plasmid was digested by NdeI and XhoI. Both the digested fragments and amplified products were analyzed by 1% agarose gel electrophoresis (Fig.3). DNA sequence analysis also indicated that the recombinant plasmid was constructed successfully.

Fig.3  Restriction enzymes digestion of recombinant pET-OVA-linker-b2m

1,  1 kb marker; 2,  pET-OVA-linker-b2m/XhoI; 3,  pET-OVA-linker-b2m/XhoI+NdeI; 4,  100 bp marker; 5,  PCR product of OVA-linker-b2m.

2.3  Analysis of the OVA-linker-b2m protein expression

After induction of the recombinant strains, the supernatant and cells were harvested and analyzed by SDS-PAGE (Fig.4) and Western blot (Fig.5). The specific band was about 12 kD. The percentage of specific protein in supernatant and cells were 1% and 46% respectively. There was only one band in buffer E,  its size being 12 kD, its concentration being 1.8 g/L.

Fig.4  Expression and purification of pET-OVA-linker-b2m

1,  crude supernatant of induced recombinant after ultrasonic wave; 2,  total protein of induced recombinant before purification;  3,  marker:  low level molecular mass; 4,  sample in buffer C after purification; 5,  sample in buffer D after purification; 6,  sample in buffer E after purification; 7,  sample in buffer B after purification.

Fig.5  Western blot analysis of insoluble expression product of pET-OVA-linker-b2m gene

1,  uninduced insoluble expression product; 2,  marker:  low level molecular mass; 3,  induced insoluble expression product.

2.4  Detection of specific epitope density by FACS

From figure 6,  it can be seen that the recombinant protein produced in E.coli permited CTL target structure formation through exogenous pathway.

Fig.6  Detection of specific epitope density by FACS

HE, LE: control HeLa-K, L929-Kb cell without fusion protein stimulation; HD: Hela-Kb cell with fusion protein stimulation; LD: L929-Kb cell with fusion protein stimulation.

2.5  Detection of CTL in vitro

C57BL/6 mice were subcutaneously injected with OVA peptide (100 mg),  OVA peptide  (2 mg)with free b2m (100 mg), OVA-linker-b2m (100 mg).7 days after priming, splenocytes were restimulated in vitro with OVA peptide（2 mmol/L）,  rhIL-2 (50 u/ml),  5 days after in vitro stimulation,  cytolytic ability of the bulk CTL was analyzed by quantitatively measuring lactate dehydrogenase (Fig.7). Spleen cells from mice treated with OVA-linker-b2m fusion protein showed a strong lytic ability against target cells; spleen cells from mice treated with OVA peptide with free b2m protein showed a weak lytic ability; spleen cells from mice treated with OVA peptide alone displayed no lytic ability following the same in vitro stimulation. The results from H-2Kb tetramer staining were consistent with cytotoxicity assay (Fig.8). Moreover,  the levels of extracellular IFN-g in these cell groups were different (Fig.9). OVA-linker-b2m group was much higher （226 ng/L）than the OVA peptide group (10 ng/L). Compared with immunizing mice with OVA peptide alone,  or using them together as immunogens,  the epitope-linked b2m appeared to be efficient, sensitive and specific in CTL induction.

Fig.7  Results from cytotoxicity assay

●,OVA peptide; ○, OVA peptide+b2m; △， OVA linker b2m.

Fig.8  H-2Kb tetramer staining specific CTL

(-),  splenocytes from mice without antibody staining; A1,  immunization with OVA peptide (100 mg); B1,  immunization with OVA peptide (2 mg)+ b2m (100 mg); C1,  immunization with OVA-linker-b2m (100 mg).

3  Discussion

    The density of specific MHC/peptide complexes on cell surface can determine the degree of T cell responsiveness,  so the ability to generate high numbers of a particular MHC class  I complex could be of great value for eliciting strong CTL  responses in the context of vaccination or immunotherapy. Unfortunately,  many peptides of clinical importance have relatively low MHC binding affinity and suboptimal immunogenicity[10, 11].One potential approach to augment the surface display and immunogenicity of an epitope is to physically couple it to its presenting MHC molecule. The peptide-binding MHC class I dimer is comprised of a polymorphic 44-kD membrane bound heavy chain interacting with an invariant 12-kD soluble light chain,  b2-microglobulin.

In the previous reports,  peptide antigens have been tethered,  via flexible polypeptide linkers,  to the heavy chain of the mouse class I molecule Kd. CTLs have been induced in vivo using the fusion protein[12].  Structurally,  tethering a peptide to b2m is less demanding than coupling antigen to the heavy chain,  as the carboxyl end of  the peptide and amino terminus of b2m are positioned relatively close together[13]. Since b2m is a soluble molecule, it is amenable for use as a protein immunogen,  unlike peptide/heavy chain fusions that must be cell surface bound. Additionally,  b2m has been observed to act as an “adjuvant” for enhancing peptide-specific CTL responses in vivo,  presumably by assisting in the MHC loading of peptides,  a phenomenon that has been extensively investigated in vitro[14]. Since the concentration of free b2m in vivo is low,  the conditions in vivo may not favor peptide association with class I molecules. The inefficient formation of peptide-class I complexes could be one factor that contributes to the failure of many peptides to prime CTL immunity in vivo. This reasoning led us to investigate whether elevated levels of xenogenetic  b2m could be used to promote peptide binding to class I molecules and elicit CTL responses[5].

Recently,  a novel method to identify antigen-specific T cells during an immune response has been developed[8]. The method involves the engineering of a biotinylation signal sequence onto the C terminus of a recombinant MHC class I or II molecule which,  after complexing with a specific peptide,  is bound to avidin at a 4∶1 ratio. This results in a tetrameric peptide-MHC complex that can recognize T cell receptors on lymphocytes specific for the particular epitope. The use of tetramers provides an advantage over currently available methods,  since the assay is more rapid and permits an assessment of the total number of peptide-specific T cells in the peripheral blood without the need for in vitro manipulation. Peptide-MHC tetramers have been widely used to quantitate the accumulation of virus and bacteria-specific T cells immunity to viruses,  including HIV,  LCMV,  influenza virus,  and Epstein-Barr virus as well as several mouse and monkey viruses[15,16].

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Received: March 18, 2002    Accepted: April 23, 2002

This work was supported by a grant from the National Natural Science Foundation of China (No.30171048)

*Corresponding author:  Tel, 86-21-63846590-776881; Fax, 86-21-63842916; e-mail, [email protected]